The administration of spironolactone is known to cause a decrease in the adrenal or testicular content of the activities of steroid hydroxylases which stems from a specific destruction of the heme of cytochrome P-450 resulting in a rapid degradation of its apoproteins. When added to microsomal suspension, spironolactone can be activated to cause a destruction of P-450 and its heme without loss of its apoproteins. In vitro experiments with microsomal suspensions and radiolabelled spironolactone have indicated: (1) spironolactone is deacetylated by an esterase in adrenal and testis microsomes to deacetylspironolactone. (2) Deacetylspironolactone is activated by cytochrome P-450 resulting in the elimination and the covalent binding of the sulfur atom to microsomal protein, but not heme, (3) The in vitro destruction of the heme does not result in the opening of the porphyrin nucleus, (4) The thio group of deacetylspironolactone may form a ligand with the heme of cytochrome P-450.